tmp7712 TMP

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COPYRIGHT 2017 EDIZIONI MINERVA MEDICA
2015 EDIZIONI MINERVA MEDICA Minerva Biotecnologica 2017 June;29(2):76-88

Online version at http://www.minervamedica.it DOI: 10.23736/S1120-4826.16.01993-5
REVIEW
Diagnostic and therapeutic applications of peptide

nucleic acid in medical microbiology
Mehrdad M. MOGHADDAM 1, 2 *, Ali MIRHOSSEINI 3, Mohammad HEIAT 1,
Hamid S. RAD 3, Reza MIRNEJAD 4, Ali CHOOPANI 1
1Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran; 2National Institute of Genetic
Engineering and Biotechnology (NIGEB), Tehran, Iran; 3Applied Microbiology Research Center, Baqiyatallah University of Medical
Sciences, Tehran, Iran; 4Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
*Corresponding author: Mehrdad M. Moghaddam, Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, 19395-5487
Tehran, Iran. E-mail: mm.genetics@gmail.com
A B S TRACT
Peptide nucleic acid (PNA) is nucleic acid analog which was formed by replacing sugar-phosphate backbone with a pseudopeptide chain consti-
tuted by N-(2-aminoethyl) glycine monomers. PNAs have attractive properties such as quiet stability against nucleases and proteases, resistance
to salt concentration variations and pH changes, and they form strong complexes with complementary strands of DNA or RNA. Generally, The
biochemical properties, strong and selective binding of PNA are interested topics to stimulated research on the hybridization process and its di-
agnostic and therapeutic applications in the past two decades. Because of this attractive nature, PNA is often used in molecular biology especially
as antisense technology to inhibit gene expression and microbial cell growth with high specificity and detection of pathogenic bacteria with high
sensitivity. According to these topics, in this review we described PNAs diagnostic and therapeutic applications in medical microbiology with
relate researches and publications in each field as practical examples.
(Cite this article as: Moghaddam MM, Mirhosseini A, Heiat M, Rad HS, Mirnejad R, Choopani A. Diagnostic and therapeutic applications of peptide
nucleic acid in medical microbiology. Minerva Biotec 2017;29:76-88. DOI: 10.23736/S1120-4826.16.01993-5)
Key words: Peptide nucleic acids -- ISIS 208529probe
PNA-FISH - Scanning probe microscopy.
- PNA-antisense - PNA-antigene
M ore than three decades ago, scientists discovered

triple DNA helix in vivo. In this structure the third
strand is bound to dsDNA by a type of hydrogen bond
molecule and named it Peptide Nucleic Acid or PNA.
In this molecule the backbone incorporates units of
N-(2-aminoethyl)-N-(pyrimidine-1-acetyl)-glycine or
called Hoogsteen bond, this triple helix is clockwise and N-(2-aminoethyl)-N-(purine-9-acetyl)-glycine instead
has three grooves. Not a long time was needed to find out of ribose and phosphate groups which are bound to one
three-stranded DNAs advantages. One is the third strand another by peptide bonds. This backbone was select-
can be used as an antigen which binds to the dsDNA and ed because distances of bonds and distances between
prevents it from transcription. It can also be modified to monomers are similar to that of nucleotides in DNA
gain enzymatic activity (a DNA enzyme) as an endonu- structure (Figure 1).1, 2
clease, but due to the charge of DNA molecule and elec-
or other proprietary information of the Publisher.
trostatic repulsion, creation of triple helices is difficult. Biochemical properties of PNA

In 1991, three scientists named Egholm, Buchardt
and Nielsen overcame this obstacle by replacing sugar- PNA strands are non-charged structures which have
phosphate backbone with poly-amide. They designed a a great affinity for binding to nucleic acids. Binding of
76 Minerva Biotecnologica June 2017

not permitted. It is not permitted to remove, cover, overlay, obscure, block, or change any copyright notices or terms of use which the Publisher may post on the Article. It is not permitted to frame or use framing techniques to enclose any trademark, log

APPLICATIONS OF PEPTIDE NUCLEIC ACID IN MICROBIOLOGY MOGHADDAM
Figure 1.Structure of PNA compared to DNA.
PNA to DNA and RNA to form DNA-PNA or PNA-

RNA hybrids takes place via normal Watson-Crick hy-
drogen bonds (Figure 2). It can also bind to dsDNA by
Hoogsteen bonds to form triple helix.3 PNA is resistant
to different kinds of nucleases and proteases and due to
its great affinity for its complementary nucleic acid met
usages beyond what was expected. Figure 2.PNA Watson-Crick base paring with DNA.
Some of main characteristics of PNA are listed be-
low.4-7
PNA is DNA/RNAs artificial analog in which Melting temperature (Tm) of PNA/DNA hybrid-
sugar-phosphate group is replaced with a pseudopep- ization is much higher than DNA/DNA and DNA/RNA
tide (polyamide) to which organic bases of DNA are hybrids. Normally, for each base pair (bp) 1 C is added
attached. to Tm.
Bases are bound to polyamide through a methy- PNA is capable to form Hoogsteen triplex in pu-
lene carbonyl linkage. rine-rich regions of DNA major groove.
Despite modifications in its structure, PNA is PNAs with 13 to 17 bp can be used as probes
completely capable to bind to its specific DNA/RNA due to their strong binding to target, while DNA probes
target. must at least be 20 to 25 bp.

Due to lack of sugar-phosphate group, PNA is PNAs specificity for its complementary region is
non-charged and so its binding to DNA is much stron- much more than DNA or RNA in a way that one mis-
ger than DNA/DNA and DNA/RNA hybrids. match in PNA/DNA hybrid causes a 15 C decrease in
Vol. 29 - No. 2 Minerva Biotecnologica 77


MOGHADDAM APPLICATIONS OF PEPTIDE NUCLEIC ACID IN MICROBIOLOGY
Tm while in DNA/RNA hybrids it will have an 11 C ner; N-terminal of PNA binds to 3 end of DNA. The
decrease. other PNA strand binds to the same region by Hoogs-
Hybridization rate of PNA/DNA (RNA) is 100- teen bonds in a parallel manner. The unbound strand of
fold higher than of DNA/DNA (RNA). Because PNA DNA forms a free loop which is called D-loop.9
is uncharged, its hybridization process is not dependent Triplex invasion helix is so temperature resistant; a
on the environmental salt concentration. For instance, 10-monomer homopyrimidine PNA has a Tm higher
in hybridization of a 15 bp PNA if NaCl concentration than 85 C but its formation is so slow in physiologi-
changes from 10 mM to 1 M, Tm only changes 5 C. cal pH due to instability of Hoogsteen bonds in triplex.
PNA is resistant to nucleases and proteases be- To overcome this problem, J base which is a pseudo-
cause of its hybrid structure. cytosine is used in PNA instead of cytosine. Cytosine
PNA is resistant to pH changes while DNA depu- needs to receive a proton (H+) in N3 position to form a
rination occurs in the pH range of 4.5 to 6.5. PNA also Hoogsteen bond and this needs a low pH condition, but
resistant to temperature changes. for J base, low pH is not necessary so Hoogsteen bonds
These remarkable characteristics enable researchers form more quickly.10-12
to utilize PNA as a capable analog for DNA/RNA. In double duplex invasion, as seen in Figure 3, two
PNA strands bind to two strands of DNA separately, to
Mechanism of PNAs binding to DNA form this structure, unusual bases are used. A and T bas-
es are replaced with 2,6-diaminopurine and 2-thiourasil,
A PNA fragment, can bind to its complementary re- respectively, in this situation 50% bases should be A/T.
gion in dsDNA in four ways according to pH, salt con- studies have shown that this binding will not prevent
centration and nucleotide sequences in its structure. transcription because RNA polymerase is able to break
These four states of binding are triplex, triplex inva- such PNA/DNA binding.5, 13, 14
sion, duplex invasion and double duplex invasion (Fig-
ure 3).4, 8 PNA applications
In a basic state, PNA binds to dsDNA by Hoogsteen
bonds and forms a triple helix (triplex). Homopyrimi- One of the most important issues in biology is to de-
dine PNA monomers form a triplex invasion structure. tect a specific sequence in DNA and bind special mol-
In this state two PNA strands simultaneously, bind to a ecules to obtain certain aims. PNAs complementary to
homopurine region in DNA. One of PNA strands binds sequence of interest can be synthesized and targeted to
to DNA by Watson-Crick bonds in an antiparallel man- the sequence to perform any operation on the target.
Figure 3.Four different types of PNAdsDNA complexes.


Table I.Some PNA applications in molecular biology. in cancer diagnostics FISH probes to improve the as-
Tools for antisense and antigene therapy say performance.18 The FISH detection methods using
Antigene technologies rRNA specific PNA probes are used for identification
Antisense technologies of microorganisms. It is not easy for DNA FISH probes
Inhibition of microRNA
Tools for molecular biology and functional genomics to match with secondary structure made by rRNA, but
Probes for Northern and Southern blot PNA-FISH probes easily overcome this obstacle and
PCR clamping discriminate any differentiation.19-21 Generally, it is ide-
Enhanced PCR
Alternative splicing
ally suited to providing accurate diagnosis of bactere-
Rare enzyme cutting mia, candidemia and sepsis in the clinical microbiology
PNA probe for diagnostics and detection lab.20, 22-24 Now, the PNA-FISH is a U.S. Food and Drug
FISH probe Administration (FDA) approved commercially avail-
SNP detection
LightUp probe, Q-PNA probe etc. able method for the detection of bacteria and yeast spe-
cies directly from positive blood culture bottles (FDA
approved PNA-FISH products of AdvanDx) (Figure
Such aims are reached through PNA modification, for 4).22, 25-27
example by binding of biocomplexes of divalent metals According to available products and clinical publica-
to PNA and redox reactions, certain sequences in DNA tions, PNA-FISH data shows high level of accuracy and
can be cleaved (PNA endonuclease activity). They can performance. For example using PNA-FISH to detect
also be used as suppressors in different levels, for in- Staphylococcus aureus possesses specificity and sensi-
stance, by designing PNAs complementary to upstream tivity 98.8% and 99.6% respectively. Also for Candida
regions of a gene promoter or its mRNA, we can pre- albicans 99.3% and 100%, Lactobacillus spp. 98% and
vent expression factors to bind to gene upstream re- 100%, Gardnerella vaginalis 100% and 100%, Entero-
gions and subsequently prevent gene expression. Some coccus faecalis 96.3% and 98.3%, and other enterococ-
proteins or other factors which take part in expression ci 93.1% and 99.3%.27, 28 Similar to this approach, in a
regulation detect specific sequences and bind to them; study by Mali et al.,20 16S rRNA specific PNA-FISH
PNAs complementary to these regions can bind specifi- probes was used for detection and identification of spe-
cally to these factors and inactivate them. So this ability cific bacteria in wound biofilms. For this purpose, PNA-
of PNA can be widely used in gene therapy and treat- FISH was used in combination with confocal laser scan-
ment of viral diseases and cancers or use as antibacterial ning microscopy (CLSM) to detect and characterize the
agents. Other applications of PNA can be listed as map- spatial distribution of biofilm-forming bacteria includ-
ping long gene sequences, point mutation, detection and ing Pseudomonas aeruginosa, Staphylococcus aureus,
accessing intra-chromosomal sequences (Table I).15-17 Streptococcus spp. and Micrococcus spp. which are pre-
dominate within human chronic skin wounds. Based on
Tools for microbial diagnostics and detection their report, by continue application of this technique
to clinical biofilms from infected tissues or indwelling
PNA-FISH probes
medical devices can be facilitated the identification and
Use as complementary nucleic acid probes is the most estimation of the relative proportions of bacteria within
common application of PNA molecules. Like common a biofilm. Also, this approach could be used to assess
nucleic acid probes, the sequences of PNA probes ex- biofilm management strategies, bacterial biofilm forma-
hibit the specificity of binding to complementary tar- tion or evaluate the effectiveness of antimicrobial agents
gets, but the sensible benefit of PNA molecule is the against members of the biofilm consortium (Figure 5).
uncharged backbone which greatly increases the probe Accordantly, PNA-FISH can be used to differentiate
affinity, PNA molecules are also stable against DNase closely related species. Hongmanee et al. applied PNA-
and proteinase. FISH to distinguish between tuberculous and non-tu-

Nowadays a wide variety of commercial PNA probe berculous Mycobacterium species in bacteria culture.29
are available for fluorescent in situ-hybridization (FISH) They reported that all PNA probes used in their study
assays. Newly, PNA molecules have been employed had high sensitivity (98) and specificity (100%). in

80
C
A
PNA staining (overlay of A-D) (from Malic et al.20).

Minerva Biotecnologica
Figure 4.Rapid identification of E. coli and P. aeruginosa from positive blood cultures by PNA-FISH probes.
biofilms labelled using three PNA probes: A) universal bacterial probe; B) P. aeruginosa-specific probe; C) S. aureus-specific probe; D) multiplex
Figure 5.Detection and identification of P. aeruginosa and S. aureus bacteria in wound biofilms by specific PNA-FISH probes. Mixed bacterial
June 2017

opsy specimens. Generally resistance to clarithromycin

can occur by several mechanisms but clarithromycin
resistance in this bacterium is almost always associated
with point mutations in the peptidyl transferase region
encoded by the V domain of the H. pylori 23S rRNA
gene which can be addressed by three described FISH
probes include: ClaR1, ClaR2, and ClaR3.33 According
to their results the PNA-FISH method was in full agree-
ment with PCR-sequencing for all regions so PNA-FISH
can provide a quick technique with high accuracy and
specificity to detection of pathogenic bacteria similar
H. pylori with both susceptible and resistant genotypes
on gastric biopsy specimens simultaneously with histol-
ogy tests. On the other hand, in 2011 Rasmussen et al.
reported a rapid assay based on PNA-FISH for iden-
A tification and determination of methicillin-resistance
coagulase negative Staphylococcus aureus (MR-CNS)
in blood cultures. In their method, they used a mecA
PNA-FISH-targeting mecA messenger RNA (mRNA)
in staphylococci performed in parallel with S. aureus/
CNS PNA-FISH for rapid identification of MRSA,
MSSA, MR-CNS, and MS-CNS within 2 hours.
In order to construction of PNA probe with high de-
gree of efficiency and different applications it should
be conjugated with different agents. For example in
so-called light-up PNA probes the cyanine dye thiazole
orange (TO) be used as uorescent conjugation label.
Upon hybridization of light-up PNA probe to DNA tar-
get, the florescence light might increase 50-folds higher
than free probes.34 Base on this approach, Lee et al. ap-
plied a detection method for Escherichia coli O157 by
B PNA array and targeting GDP-perosamine synthetase
Figure 6.Visualization of Mycobacterium tuberculosis by specific gene (per gene). This gene is specific for E. coli O157
PNA-FISH probe in a lung biopsy from an experimentally infected
mouse (from Lefmann et al.30). which contribute in formation of the O side chain in li-
popolysaccharide structure. Bacteria detection was per-
the other similar study, Lefmann et al.30 evaluated the formed based on biotin-labeled target DNA-PNA hy-
PNA-FISH for identification of clinically Mycobacteria bridization in array buffer containing Cy5-streptavidin.
including members of the M. tuberculosis complex, M. In this study the detection limit of the E. coli O157 PNA
avium, M. kansasii, and M. leprae in smears and tissue array was 100 CFU/mL.35
biopsies. The PNA probes for all samples showed 100% Quencher-labeled peptide nucleic acid (Q-PNA)
sensitivity and specificity for each Mycobacterium (Fig- probe is another example of conjugated PNA which
ure 6). plays central role in self reporting PCR. This probe
PNA-FISH is a cheap and useful tool for the iden- quenches the florescent label primers before amplifica-
tification of antibiotic resistance bacterial pathogens.31 tion so it can report negative or positive result of PCR in
Cerqueira et al.32 evaluated PNA-FISH method as a moment. Q-PNA based multiplex PCR can be designed
new diagnostic test for Helicobacter pylori clarithromy- for rapid and sensitive detection of several microbial
cin resistance detection in paraffin-embedded gastric bi- pathogens.36 For example, Xi et al. treated E. coli and


A B
C D
Figure 7.Q-PNA based multiplex PCR designed for rapid and sensitive detection of E. coli (A, B) and Methanosarcina acetivorans. Pure culture
of E. coli (A, B) and M. acetivorans (C, D) were fixed and incubated with DNA molecular beacon Bact0338 (compliment to E. coli 16S RNA; A,
C) and DNA molecular beacon Arch0915 (compliment to M. acetivorans 16S RNA; B, D) (from Xi et al.37).
Methanosarcina acetivorans bacteria cells with species- gle base mismatch will separate wild type from mutant.
specific molecular beacons in a fixed culture. A fluores- In an applicable example, it can be mentioned to rare
cein-labeled molecular beacon was specific for E. coli mutant mediated cancerous cells that may be existed be-
16S rRNA and a tetramethylrhodamine-labeled beacon tween other cells in a tissue biopsy. In PCR procedure
was specific for M. acetivorans 16S rRNA. They could using PNA clamp wild type complementary sequence,
differentiate the two bacteria species by color, generat- the amplification of wild type sequence will be blocked
ing either a green or an orange signal only for the cor- but the amplification of mutant sequence will be done
rect microorganism (Figure 7).37-39 (Figure 8).40
Peptide nucleic acid mediated PCR clamping was in-
PNA PCR clamping troduced as a powerful approach to selectively amplify
ribosomal DNAs (rDNAs) which are not frequently
PNA PCR clamping strategy was discovered in re- found in clone libraries generated by standard PCR from
cent years to fined single nucleotide polymorphism complex microbial, so by using PNA mediated PCR-
(SNP) in low frequency rate. Specific hybridization of clamping that target conserved regions of the 16S rDNA
PNA clamp with wild type sequences will arrest the sequences can be determined unknown bacterial or mi-
PCR procedure through competing with DNA primer, crobial diversity.41 In some studies also this method was
while just a base mismatch is sufficient to break PCR used for detection of bacterial pathogens, for example
blockade. So in the PNA PCR clamping technique a sin- Takiya et al. developed a rapid identification system


Figure 8.PNA-mediated real-time polymerase chain reaction (RT-PCR) clamping technology. The PNA oligomer designed to bind only to the
wild-type sequence, while the forward PCR primer acts as the competitor. A) A PNA/DNA hybrid with a perfect match prevents annealing of the
PCR primer and amplification of wild-type DNA. B) A PNA/DNA hybrid with a single base-pair mismatch will be unable to suppress annealing of
the PCR primer or amplification of mutant alleles.
of E. coli O157:H7 using PNA mediated PCR clamp- tode and found surprisingly that its prohibitory effect
ing. They confirmed a single nucleotide alteration in the is much more than when any of the strands are injected
uidA gene (-glucuronidase gene), which is specific to separately. Fire et al. called this phenomenon RNA in-
E. coli O157:H7, therefore PNA has specifically inhib- terference (RNAi). According to related studies Key
ited the PCR amplification from a wild type uidA gene.42 factors in this process are two enzymes, namely Dicer
Also Iwamoto et al. described the development of a and RNase H, RNA-induced silencing complex (RISC)
PNA-mediated competitive PCR clamping (PMCPC) and a small RNA called small interfering RNA (siRNA).
technique for the detection of mutations in the rifampin- It is noteworthy that RNAi is a natural mechanism
resistance-determining region of rpoB gene in Myco- for selective gene silencing (gene knockout) in the cells;
bacterium tuberculosis with high sensitivity for when hence it is functional in organisms bearing such pro-
resistant bacteria are present as subpopulations among cesses. Since prokaryotic cells such as bacteria do not
predominantly susceptible tubercle bacilli.43 Accord- have molecular tools for such processes, so RNAi can-
ingly, PCR clamping is done with wild-type comple- not be used to suppress bacterial genes and therefore
mentary PNA probes, so the mutant allele are selec- PNA as an alternative method with powerful potential
tively amplify and can easily be characterized by the was used for this purpose.45-47
presence of relate amplicon. By this method can be de-
tected antibiotic-resistance microorganisms with high Inhibition of transcription and translation
specificity, sensitivity in subpopulation.
The same procedure is used to enhance PCR per- PNA antisense is another application of PNA mole-
formance for exact amplification of special sequence. cules. Antisense activity is performed by natural or syn-
Since PNA weakly tolerates mismatch base paring it thetic molecules which have complementary sequences
can be used to block non-specific primer annealing by with target RNAs and block its functions. PNAs with
hybridization with primer similar sequences along the antisense sequences are able to bind to mRNA comple-
target. Using this trick the subsidiary amplification is mentary sequence and regulate its functional process.
decreased and PCR efficiency is enhanced.44 These molecules show antisense function at nano-molar
concentrations.48, 49 PNA can also play a role as anti-
Tools for antisense and antigene therapy gene agent since it can disassemble DNA double strand
structure and create PNA/DNA duplexes or triplex.50, 51
In 1998, Fire et al. injected sense and antisense PNAs with DNA mimic activity which represent a pow-
strands of an RNA sequence into the body of a nema- erful and specific antigene activity and create tight


A B C
Figure 9.Principles of the antisense and antigene gene targeting strategies. A) In normal cells, DNA is transcribed into RNA: which then is
translated into protein. B) Treatment of the cells with an antigene oligomer complementary to a specific sequence in the DNA leads to inhibition of
transcription of the gene into mRNA. C) When the cells are treated with an antisense oligomer, hybridization to a specific mRNA sequence inhibits
expression of the protein at the level of translation.
hybrids with perfect complementary DNA sequences and optimized antisense peptide-PNA conjugates tar-
are useful tools to suppress cancer-associated genes or geting the translation initiation region of the ftsZ gene
virulence genes in bacteria pathogens (Figure 9).52-54 In (an essential bacterial gene involved in cell division)
2004, Nekhotiaeva et al. was tested the growth inhibi- and the acpP gene (an essential bacterial gene involved
tory potential of antisense PNAs by targeting essential in fatty acid synthesis) of P. aeruginosa (PA01) as anti-
S. aureus growth genes. They designed PNAs to bind microbial compounds. Designed PNAs exhibited com-
within the start codon region of two well-known es- plete growth inhibition of P. aeruginosa strains PA01,
sential genes, fmhB, involved in cell wall biosynthesis, PA14, and LESB58 at 1-2 M concentrations.58
and gyrA, involved in DNA replication. In both cases, Related studies showed that some of the PNAs target-
was observed a dose-dependent inhibition of S. aureus ed to Brucella genes involved in DNA (polA, dnaG and
growth in Mueller Hinton Broth with antisense PNAs.55 gyrA), RNA (rpoB), cell envelope (asd), fatty acid (kdtA
In another other study by Kurupati et al. (2007), anti- and acpP) and protein synthesis (tsf) inhibit the growth
sense PNAs was targeted at two essential genes, gyrA of Brucella suis in culture and in macrophages after 24
and ompA, in multi-resistant -lactamase-producing hours of treatment.59 Similar study demonstrated the
Klebsiella pneumoniae strain. They demonstrated that potential of anti-rpoA (RNA polymerase subunit)
antisense PNAs were able to inhibit the growth of this PNA as an antibacterial agent to targeting intracellular
bacterium.56 Also in 2012, Bai et al. were developed pathogens like Listeria monocytogenes.60
two PNAs to target rpoD gene, which encodes an RNA
polymerase primary (70) that is thought to be essen- Inhibition of natural antisense RNAs in bacteria
tial for bacterial growth, against different clinical iso-
lates of MDR Escherichia coli, Salmonella enterica, Natural antisense RNAs (asRNA) are non-coding
Klebsiella pneumoniae, and Shigella flexneri in vitro RNAs in bacteria which are as trans and cis-antisense
and in infection models. Their results were associated sequences that regulate gene expression at the posttran-
with suppression of rpoD mRNA and (70) expression, scriptional level. Generally, asRNAs that are cis-encod-
as well as (70) downstream regulated genes including ed share high degrees of complementarity with the tar-
ftsZ, mazF, prfB, rpoS, seqA, turfB and ygjD.57 get mRNA while asRNAs that are trans-encoded have
On the other hand, Ghosal and Nielsen in 2012 intro- limited complementarity with the target mRNA, these
duced antisense peptide-PNA conjugates as antibacte- asRNAs are encoded at genomic locus distant from the
rial agents against P. aeruginosa. They were designed mRNA.47, 61, 62 asRNAs regulate some biologic process-


es such as plasmid maintenance (R1), cell proliferation, PNA transfer to target cell
host killing. So using antisense complementary oligonu-
cleotide is a simple and efficient method for inhibition In order to boost the cellular delivery of PNA in anti-
of asRNA function. PNA as an unusual oligonucleotide sense or antigene applications, it should be conjugated
with nuclease resistant properties is a potent tool to act with cell-permeable peptides or special hydrophobic
as anti-asRNA agent under various conditions.52, 63, 64 molecules since uncharged PNA pass so slowly through
Based on this future, Faridani et al. designed and used cell membrane barrier.48
anti Sok peptide nucleic acid (PNA) oligomer for block- Generally, PNA oligomers mainly have hydrophilic
ing and sequestration of Sok-RNA activity in order to characteristics and like hydrophilic peptides are not eas-
assessment of a novel antimicrobial strategy. Generally, ily taken up by prokaryotic or eukaryotic cells. So, a
among the antisense transcripts, RNA antitoxins that delivery system is needed to transfer them into the tar-
inhibit toxin mRNA translation are the best model for get cell.
mRNA-asRNA interaction studies. In E. coli the hok/ Different systems have been developed to do so, and
sok locus of plasmid R1 is an established model for according to imposing or not imposing changes in PNA
RNA antitoxin action. Interaction between hok mRNA structure, delivery systems are categorized in two main
and Sok antisense-RNA increases plasmid maintenance groups.67, 68
through post-segregational-killing of plasmid-free
progeny cells. The results showed that in E. coli cells Delivery systems without changes in PNA structure
that carrying base-pairing hok/sok, anti Sok PNA was
more bactericidal than rifampicin. Also, anti Sok PNA These systems included microinjection, electropora-
induced ghost cell morphology and an accumulation of tion, co-transfection with DNA, permeabilized cells and
mature hok mRNA, consistent with cell killing through direct delivery.
synthesis of Hok protein. It is noteworthy that based on Microinjection and electroporation approaches,
BLAST analyses many enteric bacteria have multiple moreover than application restrictions, cause damage to
hok/sok homologous and analogous RNA-regulated cells and may lead to cell destruction.
toxin-antitoxin loci.65 One of appropriate approaches of transferring mol-
ecules such as PNA without changing the structure is
cationic liposomes. But, as PNAs are not charged their
Tools for molecular biology and functional genomics transfer rate into the liposome is not so high. To im-
prove this rate, a small region of PNA is hybridized
An exact and rapid alternative Southern (Northern) with a small charged nucleotide and this charge leads
blotting is PNA Pre-Gel hybridization which is suit- the PNA/DNA hybrid into the liposome. This way the
able for analysis of small fragment of DNA like PCR PNA molecule is co-transferred with DNA into the
products and plasmids. This method is upon the special cell by liposome.
ability of PNA to bind to DNA at the presence of salt Along with these, without changing PNA structure,
with low concentration. A labeled PNA plays a central cell membrane can be permeabilized to PNA by com-
role in this procedure so that the mixture of PNA probe pounds such as streptolysin-O. Of course, PNA can be
and double stranded DNA in a low salt concentration used without delivery systems, but this requires high
solution is heated together and thereafter loaded onto an concentrations of PNA.
agarose-gel and electrophoresed. Uncharged backbone
of PNA inhibits the entrance of unboned PNA probes Delivery systems with changes in PNA structure
into the gel. Electrophoresis procedure will separate
DNA fragment with different size then the standard With some modifications in PNA structure, their de-
methods indicate the absence or presence of the labeled livery to target cells can be facilitated, for this purpose
PNA probe. PNA Pre-Gel hybridization deletes inter- a facilitating agent is added to one end of PNA strand
mediate steps that are necessary in the conventional (conjugation). Based on this purpose peptide nucleic
Southern blotting method. Several PNA probe can be acid can be conjugated to some agents including:8, 68, 69
used simultaneously in a single gel.66 conjugation to lipophilic moieties;


conjugation to peptides; Conclusions

conjugation to cell-specific receptor ligands.
The biochemical properties, strong and selective
binding of PNA stimulated research on the hybridiza-
Delivery using lipophilic compounds tion process and its therapeutic and diagnostic applica-
tions in the past two decades. More novel applications
Lipophilic compounds are a group of these facilitat-
are coming and known applications such as FISH and
ing agents. Two examples of these compounds are used
PCR clamping are becoming commercial products.
in PNA delivery studies are adamantyl acetic acid and
More diagnostic products will surely come and thera-
triphenyl phosphonium cation. Due to electrical charge
peutic products are being actively sought. However, it is
of these molecular and also potential differences in the
remarkable that antisense strategies have been consid-
membrane of target cells, triphenyl phosphonium cat-
ered as antibacterial agents by relatively few research-
ion has a great tendency to connection with cell mem-
ers, and usually as bactericidal agents targeting essential
brane and transferring into the cell; so that its concen-
genes. According to related studies antisense peptide
tration in the cell is 5 to 10 fold more than outside of
nucleic acids (PNAs) as a powerful tool can specifically
the cell.70-72
inhibit bacteria gene expression and growth, so as an
antimicrobial and anti-infective is notable.
Delivery using peptides
Considerable effort has been invested in exploring References
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Conflicts of interest.The authors certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.
Acknowledgments.The authors wish to thank their colleagues Dr Ramin Karimian and Vahab Piranfar at the Applied Biotechnology Research Center for
their kind help in preparing some of the figures.
Manuscript accepted: June 17, 2015. - Manuscript received: June 3, 2015.

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2015 EDIZIONI MINERVA MEDICA Minerva Biotecnologica 2017 June;29(2):76-88

Diagnostic and therapeutic applications of peptide

M ore than three decades ago, scientists discovered

trostatic repulsion, creation of triple helices is difficult. Biochemical properties of PNA

76 Minerva Biotecnologica June 2017

Figure 1.Structure of PNA compared to DNA.

PNA to DNA and RNA to form DNA-PNA or PNA-

target. must at least be 20 to 25 bp.

Vol. 29 - No. 2 Minerva Biotecnologica 77

Figure 3.Four different types of PNAdsDNA complexes.

78 Minerva Biotecnologica June 2017

and proteinase. FISH to distinguish between tuberculous and non-tu-

Vol. 29 - No. 2 Minerva Biotecnologica 79

PNA staining (overlay of A-D) (from Malic et al.20).

opsy specimens. Generally resistance to clarithromycin

Vol. 29 - No. 2 Minerva Biotecnologica 81

82 Minerva Biotecnologica June 2017

Vol. 29 - No. 2 Minerva Biotecnologica 83

84 Minerva Biotecnologica June 2017

Vol. 29 - No. 2 Minerva Biotecnologica 85

conjugation to peptides; Conclusions

triplexes, and quadruplexes are stabilized with trans-cyclopentane

86 Minerva Biotecnologica June 2017

Vol. 29 - No. 2 Minerva Biotecnologica 87

88 Minerva Biotecnologica June 2017

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